Article title: Transcriptional profiling efficacy to define biological activity similarity for cosmetic ingredients' safety assessment based on next-generation read-across.

The objective of this work was to use transcriptional profiling to assess the biological activity of structurally related chemicals to define their biological similarity and with that, substantiate the validity of a read-across approach usable in risk assessment. Two case studies are presented, one with 4 short alkyl chain parabens: methyl (MP), ethyl (EP), butyl (BP), and propylparaben (PP), as well as their main metabolite, p-hydroxybenzoic acid (pHBA) with the assumption that propylparaben was the target chemical; and a second one with caffeine and its main metabolites theophylline, theobromine and paraxanthine where CA was the target chemical. The comprehensive transcriptional response of MCF7, HepG2, A549 and ICell cardiomyocytes was evaluated (TempO-Seq) after exposure to vehicle-control, each paraben or pHBA, CA or its metabolites, at 3 non-cytotoxic concentrations, for 6 h. Differentially expressed genes (FDR ≥0.05, and fold change ±1.2≥) were identified for each chemical, at each concentration, and used to determine similarities. Each of the chemicals is able to elicit changes in the expression of a number of genes, as compared to controls. Importantly, the transcriptional profile elicited by each of the parabens shares a high degree of similarity across the group. The highest number of genes commonly affected was between butylparaben and PP. The transcriptional profile of the parabens is similar to the one elicited by estrogen receptor agonists, with BP being the closest structural and biological analogue for PP. In the CA case, the transcriptional profile elicited of all four methylxanthines had a high degree of similarity across the cell types, with CA and theophylline being the most active. The most robust response was obtained in the cardiomyocytes with the highest transcriptional profile similarity between CA and TP. The transcriptional profile of the methylxanthines is similar to the one elicited by inhibitors of phosphatidylinositol 3-kinase as well as other kinase inhibitors. Overall, our results support the approach of incorporating transcriptional profiling in well-designed in vitro tests as one robust stream of data to support biological similarity driven read-across procedures and strengthening the traditional structure-based approaches useful in risk assessment.

Incorporation of in vitro techniques for botanicals dietary supplement safety assessment - Towards evaluation of developmental and reproductive toxicity (DART).

As complex mixtures, botanicals present unique challenges when assessing safe use, particularly when endpoint gaps exist that cannot be fully resolved by existing toxicological literature. Here we explore in vitro gene expression as well receptor binding and enzyme activity as alternative assays to inform on developmental and reproductive toxicity (DART) relevant modes of action, since DART data gaps are common for botanicals. Specifically, botanicals suspected to have DART effects, in addition to those with a significant history of use, were tested in these assays. Gene expression changes in a number of different cell types were analysed using the connectivity mapping approach (CMap) to identify modes of action through a functional read across approach. Taken together with ligand affinity data obtained using a set of molecular targets customised towards known DART relevant modes of action, it was possible to inform DART risk using functional analogues, potency comparisons and a margin of internal exposure approach.

Use of connectivity mapping to support read across: A deeper dive using data from 186 chemicals, 19 cell lines and 2 case studies.

We previously demonstrated that the Connectivity Map (CMap) (Lamb et al., 2006) concept can be successfully applied to a predictive toxicology paradigm to generate meaningful MoA-based connections between chemicals (De Abrew et al., 2016). Here we expand both the chemical and biological (cell lines) domain for the method and demonstrate two applications, both in the area of read across. In the first application we demonstrate CMap's utility as a tool for testing biological relevance of source chemicals (analogs) during a chemistry led read across exercise. In the second application we demonstrate how CMap can be used to identify functionally relevant source chemicals (analogs) for a structure of interest (SOI)/target chemical with minimal knowledge of chemical structure. Finally, we highlight four factors: promiscuity of chemical, dose, cell line and timepoint as having significant impact on the output. We discuss the biological relevance of these four factors and incorporate them into a work flow.

Grouping 34 Chemicals Based on Mode of Action Using Connectivity Mapping.

Connectivity mapping is a method used in the pharmaceutical industry to find connections between small molecules, disease states, and genes. The concept can be applied to a predictive toxicology paradigm to find connections between chemicals, adverse events, and genes. In order to assess the applicability of the technique for predictive toxicology purposes, we performed gene array experiments on 34 different chemicals: bisphenol A, genistein, ethinyl-estradiol, tamoxifen, clofibrate, dehydorepiandrosterone, troglitazone, diethylhexyl phthalate, flutamide, trenbolone, phenobarbital, retinoic acid, thyroxine, 1α,25-dihydroxyvitamin D3, clobetasol, farnesol, chenodeoxycholic acid, progesterone, RU486, ketoconazole, valproic acid, desferrioxamine, amoxicillin, 6-aminonicotinamide, metformin, phenformin, methotrexate, vinblastine, ANIT (1-naphthyl isothiocyanate), griseofulvin, nicotine, imidacloprid, vorinostat, 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) at the 6-, 24-, and 48-hour time points for 3 different concentrations in the 4 cell lines: MCF7, Ishikawa, HepaRG, and HepG2 GEO (super series accession no.: GSE69851). The 34 chemicals were grouped in to predefined mode of action (MOA)-based chemical classes based on current literature. Connectivity mapping was used to find linkages between each chemical and between chemical classes. Cell line-specific linkages were compared with each other and to test whether the method was platform and user independent, a similar analysis was performed against publicly available data. The study showed that the method can group chemicals based on MOAs and the inter-chemical class comparison alluded to connections between MOAs that were not predefined. Comparison to the publicly available data showed that the method is user and platform independent. The results provide an example of an alternate data analysis process for high-content data, beneficial for predictive toxicology, especially when grouping chemicals for read across purposes.

Toward Good Read-Across Practice (GRAP) guidance.

Grouping of substances and utilizing read-across of data within those groups represents an important data gap filling technique for chemical safety assessments. Categories/analogue groups are typically developed based on structural similarity and, increasingly often, also on mechanistic (biological) similarity. While read-across can play a key role in complying with legislations such as the European REACH regulation, the lack of consensus regarding the extent and type of evidence necessary to support it often hampers its successful application and acceptance by regulatory authorities. Despite a potentially broad user community, expertise is still concentrated across a handful of organizations and individuals. In order to facilitate the effective use of read-across, this document aims to summarize the state-of-the-art, summarizes insights learned from reviewing ECHA published decisions as far as the relative successes/pitfalls surrounding read-across under REACH and compile the relevant activities and guidance documents. Special emphasis is given to the available existing tools and approaches, an analysis of ECHA's published final decisions associated with all levels of compliance checks and testing proposals, the consideration and expression of uncertainty, the use of biological support data and the impact of the ECHA Read-Across Assessment Framework (RAAF) published in 2015.

Dose- and Time-Dependent Transcriptional Response of Ishikawa Cells Exposed to Genistein.

To further define the utility of the Ishikawa cells as a reliable in vitro model to determine the potential estrogenic activity of chemicals of interest, transcriptional changes induced by genistein (GES) in Ishikawa cells at various doses (10 pM, 1 nM, 100 nM, and 10 µM) and time points (8, 24, and 48 h) were identified using a comprehensive microarray approach. Trend analysis indicated that the expression of 5342 unique genes was modified by GES in a dose- and time-dependent manner (P ≤ 0.0001). However, the majority of gene expression changes induced in Ishikawa cells were elicited by the highest dose of GES evaluated (10 µM). The GES' estrogenic activity was identified by comparing the Ishikawa cells' response to GES versus 17 α-ethynyl estradiol (EE, at equipotent doses, ie, 10 µM vs 1 µM, respectively) and was defined by changes in the expression of 284 unique genes elicited by GES and EE in the same direction, although the magnitude of the change for some genes was different. Further, comparing the response of the Ishikawa cells exposed to high doses of GES and EE versus the response of the juvenile rat uterus exposed to EE, we identified 66 unique genes which were up- or down regulated in a similar manner in vivo as well as in vitro Genistein elicits changes in multiple molecular pathways affecting various biological processes particularly associated with cell organization and biogenesis, regulation of translation, cell proliferation, and intracellular transport; processes also affected by estrogen exposure in the uterus of the rat. These results indicate that Ishikawa cells are capable of generating a biologically relevant estrogenic response and offer an in vitro model to assess this mode of action.

A strategy for safety assessment of chemicals with data gaps for developmental and/or reproductive toxicity.

Alternative methods for full replacement of in vivo tests for systemic endpoints are not yet available. Read across methods provide a means of maximizing utilization of existing data. A limitation for the use of read across methods is that they require analogs with test data. Repeat dose data are more frequently available than are developmental and/or reproductive toxicity (DART) studies. There is historical precedent for using repeat dose data in combination with a database uncertainty factor (UF) to account for missing DART data. We propose that use of the DART decision tree (Wu et al., 2013), in combination with a database UF, provides a path forward for DART data gap filling that better utilizes all of the data. Our hypothesis was that chemical structures identified by the DART tree as being related to structures with known DART toxicity would potentially have lower DART NOAELs compared to their respective repeat dose NOAELs than structures that lacked this association. Our analysis supports this hypothesis and as a result also supports that the DART decision tree can be used as part of weight of evidence in the selection of an appropriate DART database UF factor.

A novel transcriptomics based in vitro method to compare and predict hepatotoxicity based on mode of action.

High-content data have the potential to inform mechanism of action for toxicants. However, most data to support this notion have been generated in vivo. Because many cell lines and primary cells maintain a differentiated cell phenotype, it is possible that cells grown in culture may also be useful in predictive toxicology via high-content approaches such as whole-genome microarray. We evaluated global changes in gene expression in primary rat hepatocytes exposed to two concentrations of ten hepatotoxicants: acetaminophen (APAP), ß-naphthoflavone (BNF), chlorpromazine (CPZ), clofibrate (CLO), bis(2-ethylhexyl)phthalate (DEHP), diisononyl phthalate (DINP), methapyrilene (MP), valproic acid (VPA), phenobarbital (PB) and WY14643 at two separate time points. These compounds were selected to cover a range of mechanisms of toxicity, with some overlap in expected mechanism to address the question of how predictive gene expression analysis is, for a given mode of action. Gene expression microarray analysis was performed on cells after 24h and 48h of exposure to each chemical using Affymetrix microarrays. Cluster analysis suggests that the primary hepatocyte model was capable of responding to these hepatotoxicants, with changes in gene expression that appear to be mode of action-specific. Among the different methods used for analysis of the data, a combination method that used pathways (MOAs) to filter total probesets provided the most robust analysis. The analysis resulted in the phthalates clustering closely together, with the two other peroxisome proliferators, CLO and WY14643, eliciting similar responses at the whole-genome and pathway levels. The Cyp inducers PB, MP, CPZ and BNF also clustered together. VPA and APAP had profiles that were unique. A similar analysis was performed on externally available (TG-GATES) in vivo data for 6 of the chemicals (APAP, CLO, CPZ, MP, MP and WY14643) and compared to the in vitro result. These results indicate that transcription profiling using an in vitro assay may offer pertinent biological data to support predictions of in vivo hepatotoxicity potential.

Effects of transplacental 17-α-ethynyl estradiol or bisphenol A on the developmental profile of steroidogenic acute regulatory protein in the rat testis.

Previous research from our laboratory has determined the transcript profiles for developing fetal rat female and male reproductive tracts following transplacental exposure to estrogens. Prenatal exposure to bisphenol A (BPA) or 17-α-ethynyl estradiol (EE) significantly affects steroidogenic acute regulatory (StAR) protein transcript levels in the developing male rat reproductive tract. The purpose of this study was to establish the intratesticular distribution and temporal expression pattern of StAR, a key gene involved in steroidogenesis. Beginning on gestation day (GD) 11, pregnant Sprague-Dawley rats were exposed daily to 10µg/kg/day EE and fetal testes were harvested at GD16, 18, or 20. Quantitative reverse transcriptase PCR (QRT-PCR) demonstrated no significant difference in StAR transcript levels present at GD16. However, at GD18, StAR transcripts were significantly decreased following exposure. Immunohistochemistry demonstrated similar StAR protein levels in interstitial region of GD16 testes and an obvious decrease in StAR protein levels in the interstitial region of GD18 testes. Moreover, starting at GD11 additional dams were dosed with 0.001 or 0.1 µg/kg/day EE or 0.02, 0.5, 400 mg/kg/day BPA via subcutaneous injections. QRT-PCR validated previous microarray dose-related decreases in StAR transcripts at GD20, whereas immunohistochemistry results demonstrated decreases in StAR protein levels in the interstitial region at the highest EE and BPA doses only. Neither EE nor BPA exposure caused morphological changes in the developing seminiferous cords, Sertoli cells, gonocytes, or the interstitial region or Leydig cells at GD16-20. High levels of estrogens decrease StAR expression in the fetal rat testis during late gestation.

Predicting developmental toxicity through toxicogenomics.

Global analysis of gene expression in target cells or tissues in response to a toxicant holds significant promise for predictive toxicology. Toxicants elicit a characteristic pattern of gene expression that is dependent on mechanism of action. These mechanism-specific transcript profiles can be used as the basis for predictive toxicology. Potential applications include prioritizing chemicals for testing and customizing testing approaches based on the chemical. Results that are useful in this predictive context can be obtained from animal or in vitro models. Gene expression analysis can also be used to elucidate the shape of the dose-response curve at exposure levels below the no observed adverse effect level, an important need in risk assessment. In this review, we will illustrate each of these points using our research on estrogen and an estrogenic mode of action as a model for how to use gene expression data in a predictive way. Although gene expression in response to estrogens is tissue, life stage, and sex specific, it is feasible to identify transcript profiles that are diagnostic of this mode of action.

The genomic response of Ishikawa cells to bisphenol A exposure is dose- and time-dependent.

A reliable in vitro model to determine the potential estrogenic activity of chemicals of interest is still unavailable. To further investigate the usefulness of a human-derived cell line, we determined the transcriptional changes induced by bisphenol A (BPA) in Ishikawa cells at various doses (1 nM, 100 nM, 10 microM, and 100 microM) and time points (8, 24 and 48 h) by comparing the response of approximately 38,500 human genes and ESTs between treatment groups and controls (vehicle-treated). By trend analysis, we determined that the expression of 2794 genes was modified by BPA in a dose- and time-dependent manner (p< or =0.0001). However, the majority of gene expression changes induced in Ishikawa cells were elicited by the highest doses of BPA evaluated (10-100 microM), while the genomic response of the cells exposed to low doses of BPA was essentially negligible. By comparing the Ishikawa cells' response to BPA vs.17 alpha-ethynyl estradiol we determined that the change in the expression of 307 genes was identical in the direction of the change, although the magnitude of the change for some genes was different. Further, the response of Ishikawa cells to high doses of BPA shared similarities to the estrogenic response of the rat uterus, specifically, 362 genes were regulated in a similar manner in vivo as well as in vitro. Gene ontology analysis indicated that BPA results in changes to multiple molecular pathways affecting various biological processes particularly associated with cell organization and biogenesis, regulation of translation, cell proliferation, and intracellular transport; processes also affected by estrogen exposure in the uterus of the rat. These results indicate that Ishikawa cells are capable of generating a biologically relevant estrogenic response after exposure to chemicals with varied estrogenic activity, and offer an in vitro model to assess this mode of action.

The genomic response of a human uterine endometrial adenocarcinoma cell line to 17alpha-ethynyl estradiol.

We have determined the gene expression profile induced by 17 alpha-ethynyl estradiol (EE) in Ishikawa cells, a human uterine-derived estrogen-sensitive cell line, at various doses (1 pM, 100 pM, 10 nM, and 1 microM) and time points (8, 24, and 48 h). The transcript profiles were compared between treatment groups and controls (vehicle-treated) using high-density oligonucleotide arrays to determine the expression level of approximately 38,500 human genes. By trend analysis, we determined that the expression of 2560 genes was modified by exposure to EE in a dose- and time-dependent manner (p

Uterine temporal response to acute exposure to 17alpha-ethinyl estradiol in the immature rat.

The rat uterus responds to acute estrogen treatment with a series of well-characterized physiological responses; however, the gene expression changes required to elicit these responses have not been fully characterized. In order to understand early events induced by estrogen exposure in vivo, we evaluated the temporal gene expression in the uterus of the immature rat after a single dose of 17 alpha-ethinyl estradiol (EE) by microarray analysis, evaluating the expression of 15,923 genes. Immature 20-day-old rats were exposed to a single dose of EE (10 microg/kg), and the effects on uterine histology, weight, and gene expression were determined after 1, 2, 8, 24, 48, 72, and 96 h. EE induced changes in the expression of 3867 genes, at least at one time point (p < or = 0.0001), and at least 1.5-fold (up- or downregulated). Specifically, the expression of 8, 116, 3030, 2076, 381, 445, and 125 genes was modified at 1, 2, 8, 24, 48, 72, or 96 h after exposure to EE, respectively (p < or = 0.0001, t-test). At the tissue and organ level, a clear uterotrophic response was elicited by EE after only 8 h, reaching a maximum after 24 h and remaining detectable even after 96 h of exposure. The uterine phenotypic changes were induced by sequential changes in the transcriptional status of a large number of genes, in a program that involves multiple molecular pathways. Using the Gene Ontology to better understand the temporal response to estrogen exposure, we determined that the earliest changes were in the expression of genes whose products are involved in transcriptional regulation and signal transduction, followed by genes implicated in protein synthesis, energy utilization, solute transport, cell proliferation and differentiation, tissue remodeling, and immunological responses among other pathways. The compendium of genes here presented represents a comprehensive compilation of estrogen-responsive genes involved in the uterotrophic response.

Design of a microsphere-based high-throughput gene expression assay to determine estrogenic potential.

Recently gene expression studies have been multiplied at an accelerated rate by the use of high-density microarrays. By assaying thousands of transcripts at a time, microarrays have led to the discovery of dozens of genes involved in particular biochemical processes, for example, the response of a tissue/organ to a given chemical with therapeutic or toxic properties. The next step in these studies is to focus on the response of a subset of relevant genes to verify or refine potential therapeutic or toxic properties. We have developed a sensitive, high-throughput gene expression assay for this purpose. In this assay, based on the Luminex xMAP system, carefully selected oligonucleotides were covalently linked to fluorescently coded microspheres that are hybridized to biotinylated cRNA followed by amplification of the signal, which results in a rapid, sensitive, multiplexed assay platform. Using this system, we have developed an RNA expression profiling assay specific for 17 estrogen-responsive transcripts and three controls. This assay can evaluate up to 100 distinct analytes simultaneously in a single sample, in a 96-well plate format. This system has improved sensitivity versus existing microsphere-based assays and has sensitivity and precision comparable with or better than microarray technology. We have achieved detection levels down to 1 amol, detecting rare messages in complex cRNA samples, using as little as 2.5 microg starting cRNA. This assay offers increased throughput with decreased costs compared with existing microarray technologies, with the trade-off being in the total number of transcripts that can be analyzed.

Gene expression changes induced in the testis by transplacental exposure to high and low doses of 17{alpha}-ethynyl estradiol, genistein, or bisphenol A.

The purpose of this study was to determine (1) the transcriptional program elicited by exposure to three estrogen receptor (ER) agonists: 17 alpha-ethynyl estradiol (EE), genistein (Ges), and bisphenol A (BPA) during fetal development of the rat testis and epididymis; and (2) whether very low dosages of estrogens (evaluated over five orders of magnitude of dosage) produce unexpected changes in gene expression (i.e., a non-monotonic dose-response curve). In three independently conducted experiments, Sprague-Dawley rats were dosed (sc) with 0.001-10 microg EE/kg/day, 0.001-100 mg Ges/kg/day, or 0.002-400 mg BPA/kg/day. While morphological changes in the developing reproductive system were not observed, the gene expression profile of target tissues were modified in a dose-responsive manner. Independent dose-response analyses of the three studies identified 59 genes that are significantly modified by EE, 23 genes by Ges, and 15 genes by BPA (out of 8740), by at least 1.5 fold (up- or down-regulated). Even more genes were observed to be significantly changed when only the high dose is compared with all lower doses: 141, 46, and 67 genes, respectively. Global analyses aimed at detecting genes consistently modified by all of the chemicals identified 50 genes whose expression changed in the same direction across the three chemicals. The dose-response curve for gene expression changes was monotonic for each chemical, with both the number of genes significantly changed and the magnitude of change, for each gene, decreasing with decreasing dose. Using the available annotation of the gene expression changes induced by ER-agonist, our data suggest that a variety of cellular pathways are affected by estrogen exposure. These results indicate that gene expression data are diagnostic of mode of action and, if they are evaluated in the context of traditional toxicological end-points, can be used to elucidate dose-response characteristics.

Evaluation of the gene expression changes induced by 17-alpha-ethynyl estradiol in the immature uterus/ovaries of the rat using high density oligonucleotide arrays.

BACKGROUND: In a previous study, we determined the effects of 17-alpha-ethynyl estradiol (EE) on gene expression using microarrays that represented approximately 9,000 genes, which was the state of-the-art. Higher content arrays with almost double the number of genes have since become available. In order to better determine whether common sets of gene expression changes can be predictive of estrogenic activity, we have replicated the previous experiment using the more comprehensive microarray. METHODS: Immature 20-day-old Sprague-Dawley rats were exposed to 0.1, 1, and 10 microg EE/kg/day ( subcutaneously [s.c.]), for four days, dosing from postnatal day (PND) 20-23). Changes in a more comprehensive expression level of 15,923 rat annotated genes and expressed sequence tags were evaluated on PND 24. RESULTS: By comparing the response of the treatment groups versus controls using various statistical parameters, we determined that the expression of 1,394 genes showed a significant change with respect to control (p< or =0.0001), to at least one of the EE dosages. The tissues from animals exposed to 0.1 microg EE/kg/day showed changes in the expression of only 33 genes, whereas when they were exposed to 1 or 10 microg EE/kg/day, the expression of 409 and 548 genes was modified, respectively. A dose-dependent analysis indicated that 592 genes showed a robust and significant response to EE exposure (increased or decreased). Our analysis confirmed the regulation of previously identified estrogen-sensitive genes, and clearly identified novel mediators of estrogen actions, both in the uterus as well as in the ovary. CONCLUSIONS: This compendium of genes represents the largest compilation of estrogen-responsive genes that has ever been identified for the immature uterus and ovary of any species, and can be used to generate testable hypothesis to improve the understanding of the molecular pathways associated with physiological and pathophysiological responses to exposure to chemicals with estrogenic properties.

Gene expression changes related to growth and differentiation in the fetal and juvenile reproductive system of the female rat: evaluation of microarray results.

Microarrays make it possible to evaluate the responses of a major fraction of the genome in response to physiological perturbation or exogenous insult. This represents a huge advance in our ability to detect changes in gene expression that may be responsible for physiological or toxicological responses. Our laboratory is interested in the effects of estrogens on female reproductive system development and function. We have evaluated the changes in gene expression in response to estrogens in the female reproductive tract of rats during embryo/fetal development and in the juvenile rat (which is capable of mounting a uterotrophic response). The results of these experiments indicate that a number of genes (dozens to hundreds) are changed in a reproducible, dose-related manner in response to estrogens. These results have been published elsewhere; the purpose of this review is to evaluate, based on information from the literature, the potential role of selected genes on processes of cell proliferation and differentiation, and to suggest plausible relationships among these genes in eliciting responses at the tissue or organ level. We also discuss the utility of gene-expression experiments in elucidating the shape of the dose-response curve at low doses. In particular, we show that the dose-response for gene expression in the juvenile rat uterus is monotonic down to levels a few orders of magnitude below the NOEL for a uterotrophic response, suggesting that gene expression (and by inference higher order responses) do not follow patterns that are unpredictable based on response at higher dosages.

Impact of the phytoestrogen content of laboratory animal feed on the gene expression profile of the reproductive system in the immature female rat.

The effect of the dietary background of phytoestrogens on the outcome of rodent bioassays used to identify and assess the reproductive hazard of endocrine-disrupting chemicals is controversial. Phytoestrogens, including genistein, daidzein, and coumestrol, are fairly abundant in soybeans and alfalfa, common ingredients of laboratory animal diets. These compounds are weak agonists for the estrogen receptor (ER) and, when administered at sufficient doses, elicit an estrogenic response in vivo. In this study, we assessed the potential estrogenic effects of dietary phytoestrogens at the gene expression level, together with traditional biologic end points, using estrogen-responsive tissues of the immature female rat. We compared the gene expression profile of the uterus and ovaries, as a pool, obtained using a uterotrophic assay protocol, from intact prepubertal rats fed a casein-based diet (free from soy and alfalfa) or a regular rodent diet (Purina 5001) containing soy and alfalfa. Estrogenic potency of the phytoestrogen-containing diet was determined by analyzing uterine wet weight gain, luminal epithelial cell height, and gene expression profile in the uterus and ovaries. These were compared with the same parameters evaluated in animals exposed to a low dose of a potent ER agonist [0.1 microg/kg/day 17alpha-ethynyl estradiol (EE) for 4 days]. Exposure to dietary phytoestrogens or to a low dose of EE did not advance vaginal opening, increase uterine wet weight, or increase luminal epithelial cell height in animals fed either diet. Although there are genes whose expression differs in animals fed the soy/alfalfa-based diet versus the casein diet, those genes are not associated with estrogenic stimulation. The expression of genes well known to be estrogen regulated, such as progesterone receptor, intestinal calcium-binding protein, and complement component 3, is not affected by consumption of the soy/alfalfa-based diet when assessed by microarray or quantitative reverse transcriptase-polymerase chain reaction analysis. Our results indicate that although diet composition has an impact on gene expression in uterus and ovaries, it does not contribute to the effects of an ER agonist.

Toxicogenomic approach to endocrine disrupters: identification of a transcript profile characteristic of chemicals with estrogenic activity.

Public concerns have been raised in recent years over the possible adverse effects that may result from exposure to chemicals in the environment that have the potential to interfere with the normal function of the endocrine system in wildlife and humans ("endocrine disrupters"). Regulations have been established that require the testing of pesticides used in food crops and drinking water contaminants, for estrogenicity and other hormonal activities. In the United States, the U.S. EPA proposed the Endocrine Disrupter Screening Program, which consists of a Tier 1 screening battery of tests that is designed to identify chemicals capable of interacting with various hormonal systems, and different Tier 2 testing assays that are designed to verify and broaden the Tier 1 results. We identify 2 main problems with this approach: (1) the fact that the developmental stages that are the most susceptible to endocrine disruption are not represented in the screening tier, mainly because developmental effects tend to be latent, and there is no way to economically screen in developing models; and (2) the expense to screen each chemical to be included in this program. Thus, the need arises for an accurate, rapid, and cost effective method for assessing the potential endocrine activity of multiple chemicals during development. We hypothesize that the largely latent developmental effects of some endocrine disruptors are preceded by immediate changes in gene expression in the embryo and fetus. Therefore, an approach to assess the potential estrogenic (and other steroid hormonal) activity of different compounds is to identify those patterns of gene expression elicited in a tissue/organ exposed to these particular classes of chemicals. In this paper, the potential utility of such an approach for screening and better understanding of mechanism of action for specific chemicals with endocrine disrupter activities is presented, using as an example chemicals with estrogenic activity.